PROJECT SUMMARY ?-synuclein (SNCA) plays a central role in multiple synucleinopathies including Parkinson's disease (PD), dementia with Lewy Bodies (DLB) and multiple system atrophy (MSA) where it accumulates and contributes to cellular death. However, the precise mechanisms through which toxic ?-synuclein disrupts organelle dynamics and function in neurons are still unclear. Although endoplasmic reticulum (ER) dysfunction has been observed in multiple models of ?-synuclein toxicity, a role for pathogenic ?-synuclein in disrupting ER membrane tubulation and fusion dynamics has never been investigated. ER membrane tubulation and fusion are regulated by the GTPases atlastin and Rab10, while ER homeostasis is further regulated by selective autophagic ER degradation (ERphagy) via the autophagy receptor FAM134B. However, the role of pathogenic ?-synuclein on atlastin and Rab10 activity on ER tubules and on ERphagy dynamics is still unknown. In addition, whether ER membrane dynamics become defective over time and contribute to neurodegeneration in PD is also unclear. To address these questions, the experiments in Aim 1 will address the role of ?-synuclein in misregulating ER membrane tubulation and fusion dynamics and ERphagy using live cell imaging of human cell lines and neuronal models. To assess whether disruption of ER tubule dynamics is mediated by atlastin and Rab10, wildtype atlastin and constitutively active Rab10 will be expressed in models of ?- synuclein toxicity to determine if both defective ER morphology and ERphagy are rescued. In Aim 2, the relationship between ER membrane dynamics and neuronal dysfunction in PD will be investigated using neurons differentiated from iPSCs derived from PD patients harboring ?- synuclein locus (SNCA) duplication or triplication, as a robust model for investigating time- dependent changes in neuronal function. ER membrane dynamics of neurons plated on micropatterned substrates will be assessed with real-time imaging in aged cultures from both PD and isogenic control lines to determine if ER dynamics decline over time. In addition, as ER tubules are critical sites for regulating cellular homeostasis, ER tubulation dynamics will be promoted with atlastin and Rab10 expression to determine if rescuing ER morphology improves the function of other organelles and rescues neuronal death in PD patient-derived iPSC neurons. Ultimately, understanding the role of ER membrane dynamics in neurodegeneration will be critical for identifying the pathogenic mechanisms involved in synucleinopathies such as PD, as well as for advancing our knowledge of neuronal homeostasis which will be highly relevant to multiple other neurodegenerative diseases.